The chronic action of ACTH maintains optimal steroidogenic capacity in the adrenal cortex, assuring production of adrenal steroid hormones (cortisol, corticosterone and aldosterone) on demand. This process is mediated by cAMP exerting transcriptional pressure on genes encoding adrenocortical steroid hydroxylases (P450s) and related proteins. This competitive renewal proposes to determine how four homeodomain proteins binding to the predominant cAMP response sequence (CRS1) of bovine CYP17 (P450c17) interact with one another and the basal transcription machinery. Using in vitro transcription/translation, EMSA, cell transfection, in vitro transcription and mutagenesis; dimerization and transactivation domains in these homeodomain proteins will be identified along with coactivators and/or TAFs with which they interact. Adrenodoxin supports activity of mitochondrial P450s in the adrenal cortex and most other tissues. The gene encoding this 2Fe-2S protein is also regulated transcriptionally by ACTH(cAMP) and the novel protein binding to this CRS will be characterized in the same manner as CYP17 CRS1 binding proteins. This will permit comparison of cAMP-dependent transcriptional regulation of a gene predominantly expressed in steroidogenic cells (CYP17) with one expressed in both steroidogenic and nonsteroidogenic cells. The signalling pathway required for exertion of transcriptional pressure by ACTH of steroidogenic genes is cAMP- responsive and inhibited by cycloheximide (CHX). The cAMP-responsive/ CHX-sensitive locus will be identified as a means of understanding this signalling pathway. The proposed studies will establish for the first time at the biochemical level how peptide hormones from the anterior pituitary control levels of steroidogenic enzymes as well as providing detailed insight into the signalling pathway required for this process. These events are required for the biosynthesis of steroid hormones leading to reproductive capacity and metabolic homeostasis.